Methods of generating robust passive and active immune responses

ABSTRACT

The invention relates to methods of generating a robust passive and an active immune response in a subject comprising administering a neutralizing antibody and a pathogen to the subject. The invention also relates to kits and pharmaceutical compositions useful in generating a robust passive and an active immune response in a subject.

BACKGROUND

This application claims the benefit of priority of U.S. provisionalApplication No. 61/750,340, filed Jan. 8, 2013, the disclosure of whichis hereby incorporated by reference, as if written herein, in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the field of immunology. Moreparticularly, the present invention relates to improved methods forinducing immune responses due to infection by pathogenic agents bycombining passive and active immunization, and to antibodies thatenhance a subject's endogenous immunological response to a pathogen orto cancer cells.

DESCRIPTION OF RELATED ART

Immunity encompasses the ability of the human body to both tolerate thepresence of endogenous (“self”) material, and to eliminate foreign(“non-self”) material, such as infectious agents including viruses,bacteria, fungi, protozoa, etc., and can be achieved via two basicmechanisms: active immunity and passive immunity.

Active immunity is protection provided by one's own immune system, andinvolves stimulation of the immune system to produce antigen-specifichumoral (antibody) and cellular immunity. Unlike temporary passiveimmunity, active immunity is more permanent, lasting for many years, orfor a lifetime. The persistence of such long-lasting protection is knownas “immunological memory.” Following exposure of the immune system to anantigen, memory B-cells continue to circulate in the blood and reside inthe bone marrow for many years. Upon reexposure to the antigen, memoryB-cells begin to replicate and rapidly produce antibody, reestablishingprotection. Active immunity can be acquired in either of two ways:either by having the natural disease, or by vaccination. Vaccinesgenerate an immune response and prevent the infection with thecorresponding infectious pathogen.

Passive immunity is provided by introducing products into the body, suchas antibodies, produced by an animal or a human. This is usuallyachieved by injection. Passive immunity provides effective short-livedprotection that disappears within a few weeks or months due tocatabolism of the immunity-conferring product.

The immune system identifies foreign (“non-self”) substances, referredto as antigens. Antigens can be either live organisms,inactivated/attenuated organisms, or immune system-stimulating peptides,proteins, cell wall polysaccharides, etc., derived from host pathogens.The most effective antigens are generally live organisms. In response toan antigen, the immune system develops defensive antibodies (orimmunoglobulins) and specific T cells (cell-mediated immunity) thatfacilitate elimination of foreign substances, referred to as the “immuneresponse.”

There are several basic types of vaccines:

Live, unattenuated vaccines comprising a disease-causing pathogen;

Live, attenuated vaccines comprising a modified disease-causing pathogenthat retains the ability to replicate and produce immunity, but whichdoes not cause illness. Examples include vaccines containing liveviruses and bacteria. Active immunity from a live attenuated vaccine maynot develop due to interference from circulating antibody to thepathogen. Furthermore, antibody from any source can interfere with thegrowth of the vaccine pathogen, leading to a poor response, or noresponse, to the vaccine.

Inactivated vaccines comprising whole disease-causing pathogens thatcannot replicate, or various fractions of such pathogens. The latter canbe either protein-based or polysaccharide-based.

Recombinant vaccines in which structural elements of the infectiouspathogen are produced by recombinant DNA methods, in essence mimic all,or a portion, of the pathogen. Such vaccines are produced biologically,and when accompanied with an appropriate adjuvant, can produce a potentand neutralizing response. Examples of such vaccines include theHepatitis B vaccine, the meningococcus B vaccine, etc. However, attemptsto produce “subunit vaccines”, comprised of components (e.g., peptides)of viruses have been largely unsuccessful. Such examples include HSV,HIV, etc. Further, attempts to produce vaccines from DNA or RNA from theviruses have also met with little success.

In view of the foregoing, there is a need for improved methods andagents capable of generating an immunologically protective response insubjects infected by, or at risk for infection by, a pathogen.

SUMMARY

The invention is based, in part, on the discovery of potent antibodieswhich neutralize most, if not all, genetic variants of viruses and amethod of generating a passive and an active immune response in asubject by the administration of a neutralizing antibody with apathogen. Accordingly, in one aspect of the invention, the inventioncomprises methods for generating, stimulating or eliciting an activevaccine or immune response in a subject, or ameliorating or eliminatingthe severity of an infection in a subject, or limiting infection of asubject by a pathogen.

In one embodiment of the invention, the invention comprises a method ofgenerating an active vaccine response in a subject comprisingadministering a neutralizing antibody concurrently or sequentially withan antigen. The antigen could, for example, be an active wild type;unattenuated; attenuated; partially inactivated; or totally inactivatedinfectious pathogen. In another embodiment, the invention comprises amethod of stimulating an active immune response in a subject, comprisingadministering to the subject an effective amount of an antibody and anantigen, wherein the antigen is an infectious pathogen that causesdisease or is equivalent to a pathogen that causes a disease, andwherein the antibody neutralizes infectivity of the pathogen. In yetanother embodiment, the invention comprises a method of ameliorating oreliminating the severity of an infection, comprising administering to asubject in need thereof an effective amount of an antibody and anantigen, wherein the infection is caused by the antigen, wherein theantigen is an infectious pathogen that causes a disease or is equivalentto a pathogen that causes a disease, and wherein the antibodyneutralizes infectivity of the pathogen. In yet another embodiment, theinvention comprises a method of limiting infection of a subject by apathogen, comprising administering to a subject in need of suchprotection an effective amount of an antibody and an antigen, whereinthe antigen is an infectious pathogen that causes a disease or anequivalent thereof; and wherein the antibody neutralizes infectivity ofthe pathogen.

In another aspect, the invention comprises a method of ameliorating oreliminating the severity of an existing infection caused by a pathogen,comprising administering to a subject in need thereof an effectiveamount of an antibody and an antigen, wherein the antigen is an activewild type; unattenuated; attenuated; partially inactivated; or totallyinactivated infectious pathogen that causes the infection or isequivalent to the pathogen causing the existing infection, and whereinthe antibody neutralizes infectivity of either the pathogen causing theexisting infection, or the equivalent pathogen, or both. In oneembodiment the antibody neutralizes the pathogen, yet allows the naturalimmune response to the pathogen to develop, thus eliciting avaccine-like persistent immunological response, while neutralizing thepathological effects of the pathogen. In another embodiment, theantibody neutralizes infectivity of the pathogen and enhances thesubject's endogenous immunological response to the pathogen. In yetanother embodiment, the antibody neutralizes infectivity of the pathogenand allows the natural immune response process to continue at somelevel.

In yet another aspect, the invention comprises a kit that comprises anantibody and an antigen, wherein the antigen is an active wild type;unattenuated; attenuated; partially inactivated; or totally inactivatedinfectious pathogen; and wherein the antibody neutralizes infectivity ofthe pathogen.

The invention further comprises a pharmaceutical composition comprisingan antibody, an antigen, and a pharmaceutically acceptable carrier,diluent, or excipient, wherein the antigen is an active wild type;unattenuated; attenuated; partially inactivated; or totally inactivatedinfectious pathogen, and wherein the antibody neutralizes infectivity ofthe pathogen.

DESCRIPTION OF FIGURES

FIG. 1 shows the body weight loss (A to D panels) and survival (E panel)of mice (five animals per group) administered with the indicated dosesof the neutralizing antibody FI6 four hours before infection with asub-lethal challenge with H1N1 A/Puerto Rico/8/34 influenza A virus.

FIG. 2 shows the neutralizing (IC50 left part) and binding (EC50 rightpart) reciprocal antibody titers in sera of mice collected 25 days afterinfection and treatment with the indicated doses of FI6.

FIG. 3 shows the survival (A) of Syrian hamsters infected with a lethaldose of rabies virus and treated with a combination of two neutralizingantibodies at different time points after infection and the level ofproduction of endogenous anti-rabies antibodies in the differentexperimental groups (B). PEP, post-exposure prophylaxis given from dayone after infection based on rabies vaccine and 20 IU/kg ofrabies-specific immunoglobulins (RIG).

DETAILED DESCRIPTION

The invention is derived in part from the discovery of potent antibodieswhich neutralize most, if not all, genetic variants of viruses (CortiD., et al., 2011, A neutralizing antibody selected from plasma cellsthat binds to group 1 and group 2 influenza A hemagglutinins. Science,333:850-856) and the discovery of a method of generating a passive andan active immune response in a subject comprising administering aneutralizing antibody and a pathogen to the subject. The invention isfurther based on the discovery that the use of neutralizing antibodiesdoes not block the generation of immunological memory and long-lastingprotection and could, through a series of mechanisms, alter and improvethe quality of the immune response to the given pathogen.

The methods of the invention provide a combination of passive and activeimmunization. The neutralizing antibody prevents or decreasespathogen-induced symptoms without compromising the generation ofimmunological memory and long-term protection. Further, there may beadditional benefits such as a stronger and qualitatively improved, andpossibly different, immune response.

Without being bound to any theory, it is believed that the antibodyincreases presentation of the antigen to B cells and T cells. It is alsobelieved that the antibody skews T helper cell response, i.e., Th1 vsTh2 T cell responses, which could be particularly advantageous for Tcell vaccines such as vaccines against tuberculosis or RSV.

By administering a neutralizing antibody and a pathogen, neutralized bythe antibody, to a subject, the subject can get immediate protectionfrom the disease or infection caused by the pathogen via the injectionof the antibody, and broad, active immunity (both B-cell and T-cellbased immunity) in response to the pathogen. This may enable thedevelopment of a simple vaccine process, and, importantly, allowsdevelopment of vaccines for infections or disorders for which vaccineswere, until now, difficult to develop. Such infections or disorders arethose for which it is difficult to obtain the key epitopes in a formthat provokes a broad immunological response required of a vaccine.Examples of such diseases or disorders include, but are not limited to,those caused by pathogens such as human immunodeficiency virus type 1(HIV-1), human immunodeficiency virus type 2 (HIV-2), hepatitis C (HCV)etc. With the methods of the present invention, it is now possible toelicit a broad immunological response by the administration of suchpathogens as well as very potent antibodies that neutralize theinfectivity of the pathogens.

In one aspect of the invention, the invention comprises methods forgenerating, stimulating or eliciting a passive and an active vaccine orimmune response in a subject, or ameliorating or eliminating theseverity of an infection in a subject, or limiting infection of asubject by a pathogen.

In one embodiment of the invention, the invention comprises a method ofgenerating an active vaccine or immune response in a subject comprisingadministering to the subject an effective amount of an antibody and anantigen. The antigen administered is an active wild type; unattenuated;attenuated; partially inactivated; or totally inactivated infectiouspathogen that causes a disease or disorder, and the antibody is aneutralizing antibody that neutralizes infectivity of the pathogen.

In another embodiment, the invention comprises a method of amelioratingor eliminating the severity of an infection caused by an active wildtype; unattenuated; attenuated; partially inactivated; or totallyinactivated infectious pathogen, comprising administering to a subjectin need thereof, an effective amount of an antibody and an antigen. Theantigen administered is the pathogen that causes the infection ordisease or an equivalent thereof, and the antibody is a neutralizingantibody that neutralizes infectivity of the pathogen that causes thedisease, the equivalent pathogen or both.

In yet another embodiment, the invention comprises a method of limitinginfection of a subject by an active wild type; unattenuated; attenuated;partially inactivated; or totally inactivated infectious pathogen,comprising administering to a subject in need of such protection aneffective amount of an antibody and an antigen. The antigen administeredis the pathogen that causes the infection, disorder or disease, or anequivalent thereof, and the antibody is a neutralizing antibody thatneutralizes infectivity of the pathogen that causes the disease, theequivalent pathogen or both.

In one embodiment, the subject is immunologically protected from theantigenic pathogen by the active immune response, the antibody, or both.The response is, in general, robust, persistent and fully protective.The protection may, but need not be, greater than the protection seenwhen the subject is naturally infected by the pathogen and subsequentlyrecovers from such infection. In one embodiment, the protection affordedby the methods of the invention is greater than the protection seen whenthe subject is naturally infected by the pathogen and subsequentlyrecovers from such infection. In another embodiment, the two types ofprotection are comparable.

The antigen can be any wild type; unattenuated; attenuated; partiallyinactivated; or totally inactivated; but infectious pathogen, now knownto one of skill in the art, or later discovered, that causes aninfection, disease or disorder and for which neutralizing or protectiveantibodies can be obtained. In one embodiment, the antigen is a pathogennow known to one of skill in the art, or later discovered, that causes adisease or disorder for which potent and broadly protective neutralizingantibodies can be obtained.

Examples of pathogens that can be used as antigens of the inventioninclude, but are not limited to, viral, bacterial, protozoan or fungalpathogens. In one embodiment, the pathogen is a mammalian or avianpathogen. In another embodiment, the pathogen is a human pathogen. Inyet another embodiment, the pathogen is selected from the listincluding, but not limited to, human inununodeficiency virus type 1(HIV-1), human immunodeficiency virus type 2 (HIV-2), simianimmunodeficiency virus (SIV), simian-HIV (SHIV), feline immunodeficiencyvirus (FIV), hepatitis A virus, hepatitis B virus, hepatitis C virus,herpes simplex virus type 1 or type 2, SARS coronavirus , measles virus,mumps virus, rubella virus, rabies virus, ebola virus, influenza Avirus, influenza B virus, papillomavirus , rhinovirus, vaccinia virus,varicella-zoster virus , variola virus , polio virus, rhino virus,respiratory syncytial virus (RSV), metapneumovirus (MPV), dengue virus,a human endogenous retrovirus, P. falciparum, P. vivax, P. malariae, P.ovale, Corynebacterium diphtheriae, Clostridium tetani, Clostridiumbotulimum, Bordetella pertussis Haemophilus influenzae , Neisseriameningitidis, serogroup A, B, C, W135 and/or Y, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus pyogenes,Staplaylococcus aureus, Bacillus anthracis, Moraxella catarrhalis,Chlamydia trachomatis, Chlamydia pneumoniae, Yersinia pestis,Francisella tularensis, Salmonella species, Vibrio cholerae, toxic E.coli, Mycobaterium tuberculosis, Burkholderia pseudomallei and othermicrobial pathogens.

The invention also comprises a method of ameliorating or eliminating theseverity of an existing infection caused by an active wild type;unattenuated; attenuated; partially inactivated; or totally inactivatedinfectious pathogen, comprising administering to a subject in needthereof, an effective amount of an antibody and an antigen. The antigenis a pathogen that causes the existing infection, or is equivalent tothe pathogen causing the existing infection, and the antibody is aneutralizing antibody that neutralizes infectivity of either thepathogen causing the existing infection, or the equivalent pathogen, orboth.

In the case of an existing infection, the antigen administered can, butneed not be, an infectious pathogen. In one embodiment, the antigen isan active wild type; live, unattenuated; attenuated; partiallyinactivated; or totally inactivated, but infectious pathogen asdescribed above, that is equivalent to the pathogen causing the existinginfection. In another embodiment, the antigen is an attenuated orpartially or totally inactivated, or non-infectious pathogen that isequivalent to the pathogen causing the existing infection.

In yet another embodiment of the invention, the invention provides amethod of ameliorating or eliminating the severity of an existinginfection caused by an active wild type; unattenuated; attenuated;partially inactivated; or totally inactivated infectious pathogen,comprising administering to a subject in need thereof an effectiveamount of an antibody. The antibody administered enhances the subject'sendogenous or active immunological response to the pathogen andneutralizes infectivity of the pathogen. This is seen, for example, inindividuals infected with highly pathogenic viruses such as H5N1 virusand SARS virus.

In another aspect, the invention provides a kit that comprises anantibody and/or an antigen. In one embodiment, the antigen is aninfectious pathogen as described above and the antibody neutralizesinfectivity of the pathogen. In another embodiment, the antigen is anattenuated or partially or completely inactivated, or non-infectiouspathogen as described above. In yet another embodiment, the antibodyenhances the subject's endogenous or active immunological response tothe pathogen and neutralizes infectivity of the pathogen.

In yet another aspect, the invention provides a pharmaceuticalcomposition comprising an antibody and/or an antigen, and apharmaceutically acceptable carrier, diluent, or excipient. In oneembodiment, the antigen is an infectious pathogen as described above andthe antibody neutralizes infectivity of the pathogen. In anotherembodiment, the antigen is an attenuated or partially or totallyinactivated, or non-infectious pathogen as described above. In yetanother embodiment, the antibody enhances the subject's endogenous oractive immunological response to the pathogen and neutralizesinfectivity of the pathogen.

In the methods of the invention the antibody and antigen may beadministered concurrently or sequentially. In one embodiment, theantibody and antigen are administered concurrently, i.e., they areadministered simultaneously, or within a few seconds, for example,within one (1) or two (2) seconds, of each other.

In other embodiments, the antibody and antigen may be administeredsequentially. In one embodiment, the antibody is administered first,followed by administration of the antigen. In another embodiment, theantigen is administered first, followed by administration of theantibody. In yet another embodiment, the antibody and the antigen areadministered concurrently. In still another embodiment, the antibody andthe antigen are administered as an antibody-antigen complex.

In those embodiments of the invention where the antibody and antigen areadministered sequentially, i.e., antibody first, followed by antigen, orantigen first, followed by antibody, the time interval between theadministration of the first and second moieties could be as little as afew seconds, for example, two, three, four, five, or ten seconds, orcould be as large as one day or several days, for example, two, three,four, five, six, eight, ten or twelve days, or even weeks, for example,one, two, three or four weeks. In one embodiment, the time intervalranges from about 5 seconds to about 3 weeks.

In another embodiment, the time interval ranges from about 5 seconds toabout 2 weeks, or from about 5 seconds to about 10 days, or from about 5seconds to about 1 week, or from about 5 seconds to about 5 days, orfrom about 5 seconds to about 4 days, or from about 5 seconds to about 3days, or from about 5 seconds to about 2 days, or from about 5 secondsto about 36 hours, or from about 5 seconds to about 1 day, or from about5 seconds to about 18 hours, or from about 5 seconds to about 12 hours,or from about 5 seconds to about 8 hours, or from about 5 seconds toabout 6 hours, or from about 5 seconds to about 4 hours, or from about 5seconds to about 2 hours, or from about 5 seconds to about 1 hour, orfrom about 5 seconds to about 45 minutes, or from about 5 seconds toabout 30 minutes, or from about 5 seconds to about 20 minutes, or fromabout 5 seconds to about 15 minutes, or from about 5 seconds to about 10minutes, or from about 5 seconds to about 5 minutes, or from about 5seconds to about 3 minutes, or from about 5 seconds to about 1 minute,or from about 5 seconds to about 30 seconds.

In yet another embodiment, the time interval ranges from about 10seconds to about 3 weeks, or from about 15 seconds to about 3 weeks, orfrom about 30 seconds to about 3 weeks, or from about 45 seconds toabout 3 weeks, or from about 1 minute to about 3 weeks, or from about 2minutes to about 3 weeks, or from about 3 minutes to about 3 weeks, orfrom about 5 minutes to about 3 weeks, or from about 7 minutes to about3 weeks, or from about 10 minutes to about 3 weeks, or from about 15minutes to about 3 weeks, or from about 20 minutes to about 3 weeks, orfrom about 30 minutes to about 3 weeks, or from about 45 minutes toabout 3 weeks, or from about 1 hour to about 3 weeks, or from about 2hours to about 3 weeks, or from about 3 hours to about 3 weeks, or fromabout 4 hours to about 3 weeks, or from about 6 hours to about 3 weeks,or from about 8 hours to about. 3 weeks, or from about 12 hours to about3 weeks, or from about 18 hours to about 3 weeks, or from about 1 day toabout 3 weeks, or from about 36 hours to about 3 weeks, or from about 2days to about 3 weeks, or from about 3 days to about 3 weeks, or fromabout 4 days to about 3 weeks, or from about 7 days to about 3 weeks, orfrom about 1 week to about 3 weeks, or from about 2 weeks to about 3weeks.

in yet another embodiment, the time interval ranges from about 10seconds to about 2 weeks, or from about 15 seconds to about 10 days, orfrom about 30 seconds to about 10 days, or from about 1 minute to about10 days, or from about 2 minutes to about 10 days, or from about 5minutes to about 10 days, or from about 10 minutes to about 10 days, orfrom about 20 minutes to about 10 days, or from about 30 minutes toabout 10 days, or from about 1 hour to about 10 days, or from about 2hours to about 10 days, or from about 4 hours to about 10 days, or fromabout 6 hours to about 10 days, or from about 8 hours to about 10 days,or from about 12 hours to about 10 days, or from about 18 hours to about10 days, or from about 24 hours to about 10 days, or from about 36 hoursto about 10 days, or from about 2 days to about 10 days, or from about 3days to about 10 days, or from about 4 days to about 10 days, or fromabout 5 days to about 10 days, or from about 7 days to about 10 days.

The time interval between the administration of the antibody and theantigen, or the antigen and the antibody, could be important inoptimizing the effect. This time interval will depend on severalfactors, such as the dosage of the antibody and the antigen, the degreeof infectivity of the antigenic pathogen, the route of administration ofthe antibody and the antigen, the immune system of the subject etc. Oneof skill in the art would have the knowledge to adjust the time intervalbased on these factors in order to ascertain the optimum effect.

In addition to the time interval, the dose or quantity or concentrationof the antibody and antigen is also important. As described in Example 1below, the doses of the antibody and/or the antigen need to becontrolled such that the subject is protected against infection by thepathogen, while allowing the body to generate the endogenous immuneresponse. The antibody should not completely interfere with replicationof the pathogen. The right balance will reduce the infectivity (andsymptoms) without completely abolishing the generation of antigenicmaterial necessary to elicit the immune response. The dose should beestablished case by case, but the principle to be followed is that theantibody dose and administration schedule should be tuned to not conferfull neutralization or blocking of the pathogen replication.

The dose of antibody administered will depend on several factorsincluding, but not limited to, the potency of the antibody, the route ofadministration as discussed below, the immune system of the subject,whether the antibody is being administered before, or after, theantigen, etc. One of skill in the art would have the knowledge to adjustthe dose of the antibody based on these factors. In one embodiment, theantibody is administered at doses from about 0.01 mg/kg (10 ng/kg) toabout 100 mg/kg. In another embodiment, the antibody is administered atdoses from about 0.01 mg/kg to about 75 mg/kg, or from about 0.01 mg/kgto about 50 mg/kg, or from about 0.05 mg/kg (5 ng/kg) to about 100mg/kg, or from about 0.05 mg/kg to about 75 mg/kg, or from about 0.01mg/kg to about 50 mg/kg, or from about 0.1 mg/kg to about 100 mg/kg, orfrom about 0.1 mg/kg to about 75 mg/kg, or from about 0.1 mg/kg to about50 mg/kg.

In another embodiment, the antibody is administered at doses from about0.1 mg/kg to about 45 mg/kg, or from about 0.1 mg/kg to about 40 mg/kg,or from about 0.1 mg/kg to about 35 mg/kg, or from about 0.1 mg/kg toabout 30 mg/kg, or from about 0.1 mg/kg to about 25 mg/kg, or from about0.1 mg/kg to about 20 mg/kg, or from about 0.1 mg/kg to about 15 mg/kg,or from about 0.1 mg/kg to about 10 mg/kg, or from about 0.1 mg/kg toabout 5 mg/kg, or from about 0.1 mg/kg to about 4 mg/kg, or from about0.1 mg/kg to about 3 mg/kg, or from about 0.1 mg/kg to about 2 mg/kg, orfrom about 0.1 mg/kg to about 1 mg/kg, or from about 0.1 mg/kg to about0.8 mg/kg, or from about 0.1 mg/kg to about 0.6 mg/kg, or from about 0.1mg/kg to about 0.5 mg/k, or from about 0.1 mg/kg to about 0.3 mg/kg, orfrom about 0.01 mg/kg to about 1 mg/kg, or from about 0.01 mg/kg toabout 0.8 mg/kg, or from about 0.01 mg/kg to about 0.6 mg/kg, or fromabout 0.01 mg/kg to about 0.5 mg/kg, or from about 0.01 mg/kg to about0.3 mg/kg, or from about 0.01 mg/kg to about 0.1 mg/kg.

In yet another embodiment, the antibody is administered at doses fromabout 0.2 mg/kg to about 50 mg/kg, or from about 0.5 mg/kg to about 50mg/kg, or from about 1 mg/kg to about 50 mg/kg, or from about 2 mg/kg toabout 50 mg/kg, or from about 3 mg/kg to about 50 mg/kg, or from about 4mg/kg to about 50 mg/kg, or from about 5 mg/kg to about 50 mg/kg, orfrom about 10 mg/kg to about 50 mg/kg, or from about 15 mg/kg to about50 mg/kg, or from about 20 mg/kg to about 50 mg/kg, or from about 25mg/kg to about 50 mg/kg, or from about 30 mg/kg to about 50 mg/kg, orfrom about 35 mg/kg to about 50 mg/kg, or from about 40 mg/kg to about50 mg/kg, or from about 45 mg/kg to about 50 mg/kg, or from about 30mg/kg to about 75 mg/kg, or from about 40 mg/kg to about 75 mg/kg, orfrom about 30 mg/kg to about 100 mg/kg, or from about 40 mg/kg to about50 mg/kg, or from about 50 mg/kg to about 70 mg/kg, or from about 50mg/kg to about 80 mg/kg, or from about 50 mg/kg to about 90 mg/kg, orfrom about 50 mg/kg to about 100 mg/kg, or from about 70 mg/kg to about90 mg/kg, or from about 70 mg/kg to about 100 mg/kg.

In still another embodiment, the antibody is administered at doses fromabout 1 mg/kg to about 3 mg/kg, or from about 1 mg/kg to about 5 mg/kg,or from about 1 mg/kg to about 7 mg/kg, or from about 1 mg/kg to about10 mg/kg, or from about 5 mg/kg to about 10 mg/kg, or from about 5 mg/kgto about 15 mg/kg, or from about 5 mg/kg to about 20 mg/kg, or fromabout 10 mg/kg to about 15 mg/kg, or from about 10 mg/kg to about 20mg/kg, or from about 10 mg/kg to about 25 mg/kg, or from about 10 mg/kgto about 30 mg/kg, or from about 15 mg/kg to about 20 mg/kg, or fromabout 15 mg/kg to about 25 mg/kg, or from about 15 mg/kg to about 30mg/kg, or from about 15 mg/kg to about 35 mg/kg, or from about 20 mg/kgto about 25 mg/kg, or from about 20 mg/kg to about 30 mg/kg, or fromabout 20 mg/kg to about 35 mg/kg, or from about 20 mg/kg to about 40mg/kg, or from about 25 mg/kg to about 30 mg/kg, or from about 25 mg/kgto about 35 mg/kg, or from about 25 mg/kg to about 40 mg/kg, or fromabout 25 mg/kg to about 45 mg/kg, or from about 25 mg/kg to about 50mg/kg.

Just as with the antibody, the dose of antigen administered varies fromcase to case, and will depend on several factors, including, but notlimited to, the potency of the antibody that is being administered, theinfectivity of the antigen, the route of administration as discussedbelow, the immune system of the subject, whether the antigen is beingadministered before, or after, the antibody, etc. One of skill in theart would have the knowledge to adjust the dose of the antigen based onthese factors. In one embodiment, the antigen is an active wild type;unattenuated; attenuated; partially inactivated; or totally inactivated,but infectious pathogen and is administered at doses from about 1infectious or colony forming unit to about 10¹⁰ infectious or colonyforming units.

In another embodiment, the antigen is an active wild type; unattenuated;attenuated; partially inactivated; or totally inactivated, butinfectious pathogen and is administered at doses from about 1 infectiousor colony forming unit to about 10⁹ infectious or colony forming units,or from about 1 infectious or colony forming unit to about 10⁸infectious or colony forming units, or from about 1 infectious or colonyforming unit to about 10⁷ infectious or colony forming units, or fromabout 1 infectious or colony forming unit to about 10⁶ infectious orcolony forming units, or from about 1 infectious or colony forming unitto about 10⁵ infectious or colony forming units, or from about 1infectious or colony forming unit to about 10⁴ infectious or colonyforming units, or from about 1 infectious or colony forming unit toabout 5000 infectious or colony forming units, or from about 1infectious or colony forming unit to about 1000 infectious or colonyforming units, or from about 1 infectious or colony forming unit toabout 500 infectious or colony forming units, or from about 1 infectiousor colony forming unit to about 100 infectious or colony forming units,or from about 1 infectious or colony forming unit to about 50 infectiousor colony forming units, or from about 1 infectious or colony formingunit to about 10 infectious or colony forming units, or from about 1infectious or colony forming unit to about 5 infectious or colonyforming units.

In another embodiment, the antigen is an active wild type; unattenuated;attenuated; partially inactivated; or totally inactivated, butinfectious pathogen and is administered at doses from about 10infectious or colony forming units to about 10¹⁰ infectious or colonyforming units, or from about 50 infectious or colony forming units toabout 10¹⁰ infectious or colony forming units, or from about 100infectious or colony forming units to about 10¹⁰ infectious or colonyforming unit, or from about 500 infectious or colony forming units toabout 10¹⁰ infectious or colony forming units, or from about 1000infectious or colony forming units to about 10¹⁰ infectious or colonyforming units, or from about 10⁴ infectious or colony forming units toabout 10¹⁰ infectious or colony forming units, or from about 10⁵infectious or colony forming unit to about 10¹⁰ infectious or colonyforming units, or from about 10⁶ infectious or colony forming units toabout 10¹⁰ infectious or colony forming units, or from about 10⁷infectious or colony forming units to about 10 ¹⁰ infectious or colonyforming units, or from about 10⁷ infectious or colony forming units toabout 10 ¹⁰ infectious s or colony forming units, or from about 10⁸infectious or colony forming units to about 10¹⁰ infectious or colonyforming units, or from about 10⁹ infectious or colony forming units toabout 10¹⁰ infectious or colony forming units.

In yet another embodiment, the antigen is an active wild type;unattenuated; attenuated; partially inactivated; or totally inactivated,but infectious pathogen and is administered at doses from about 10infectious or colony forming units to about 50 infectious or colonyforming units, or from about 10 infectious or colony forming units toabout 100 infectious or colony forming units, or from about 10infectious or colony forming units to about 500 infectious or colonyforming unit, or from about 10 infectious or colony forming units toabout 1000 infectious or colony forming units, or from about 50infectious or colony forming units to about 500 infectious or colonyforming units, or from about 50 infectious or colony forming units toabout 1000 infectious or colony forming units, or from about 50infectious or colony forming unit to about 5000 infectious or colonyforming units, or from about 100 infectious or colony forming units toabout 1000 infectious or colony forming units, or from about 100infectious or colony forming units to about 5000 infectious or colonyforming units, or from about 100 infectious or colony forming units toabout 10⁴ infectious or colony forming units, or from about 500infectious or colony forming units to about 5000 infectious or colonyforming units, or from about 500 infectious or colony forming units toabout 10⁴ infectious or colony forming units or from about 500infectious or colony forming units to about 10⁵ infectious or colonyforming units, or from about 1000 infectious or colony forming units toabout 10⁴ infectious or colony forming units, or from about 1000infectious or colony forming units to about 10⁵ infectious or colonyforming units, or from about 10⁴ infectious or colony forming units toabout 10⁵ infectious or colony forming units, or from about 10⁴infectious or colony forming units to about 10⁶ infectious or colonyforming units, or from about 10⁵ infectious or colony forming units toabout 10⁶ infectious or colony forming units, or from about 10⁵infectious or colony forming units to about 10⁷ infectious or colonyforming units, or from about 10⁶ infectious or colony forming units toabout 10⁷ infectious or colony forming units, or from about 10⁶infectious or colony forming units to about 10⁸ infectious or colonyforming units, or from about 10⁷ infectious or colony forming units toabout 10⁸ infectious or colony forming units, or from about 10⁷infectious or colony forming units to about 10⁹ infectious or colonyforming units, or from about 10⁸ infectious or colony forming units toabout 10⁹ infectious or colony forming units, or from about 10⁸infectious or colony forming units to about 10¹⁰ infectious or colonyforming units, or from about 10⁹ infectious or colony forming units toabout 10¹⁰ infectious or colony forming units.

In another embodiment, the antigen is an attenuated, partiallyinactivated, or non-infectious pathogen and is administered at dosesfrom about 0.1 μg to about 1000 μg, or from about 0.1 μg to about 750μg, or from about 0.1 μg to about 500 μg, or from about 0.1 μg to about250 μg, or from about 0.1 μg to about 100 μg, or from about 0.1 μg toabout 50 μg, or from about 0.1 μg to about 25 μg, or from about 0.1 μgto about 10 μg, or from about 0.1 μg to about 5 μg, or from about 0.1 μgto about 1 μg, or from about 1 μg to about 1000 μg, or from about 5 μgto about 1000 μg, or from about 10 μg to about 1000 μg, or from about 25μg to about 1000 μg, or from about 50 μg to about 1000 μg, or from about75 μg to about 1000 μg, or from about 100 μg to about 1000 μg, or fromabout 200 μg to about 1000 μg, or from about 400 μg to about 1000 μg, orfrom about 500 μg to about 1000 μg, or from about 600 μg to about 1000μg, or from about 700 μg to about 1000 μg, or from about 800 μg to about1000 μg, from about 900 μg to about 1000 μg.

The invention further contemplates the administration of multiple dosesof antibody and antigen. In one embodiment, 1, 2, 3, or more doses ofthe antibody are administered. In another embodiment, 1, 2, 3, or moredoses of the antigen are administered. The doses of antibody and antigenmay be administered concurrently or sequentially, as discusse above.Further, if the doses of antibody and antigen are administeredsequentially, they may be administered in any order, and with the timeintervals as discussed above.

The route of administration of the antibody and antigen is alsoimportant as different routes confer different rates and levels ofbioavailability as well as result in the elicitation of differentresponse mechanisms. In the methods, kits and pharmaceuticalcompositions of the invention the antibody and antigen may beadministered by either the same, or by different routes. In oneembodiment, the antibody and the antigen are administered by the sameroute. In another embodiment, the antibody and the antigen areadministered by different routes. Examples of such routes include, butare not limited to, oral, dermal, intranasal, intramuscular,intravenous, intrathecal, intravaginal, intravitreal, rectaladministration, subcutaneous, intra-arterial, intramedullary,intraperitoneal, intraventricular, transdemial, transcutaneous, topical,enteral or sublingual.

The antibody used in the methods, kits and pharmaceutical compositionsof the invention may be monoclonal or polyclonal; they may be humanantibodies or humanized antibodies. In addition, they may be antibodyfragments that have retained their ability to bind the antigen as wellas their protective/neutralizing ability. In one embodiment, theantibodies are very potent and only a low concentration of theantibodies is needed for neutralizing the pathogen. In anotherembodiment, the antibodies are very broadly neutralizing and have theability to neutralize several strains, clades, sub-groups or groups of apathogen.

The antibody may be isolated, and may be provided in purified form.Typically, the antibody will be present in a composition that issubstantially free of other polypeptides e.g., where less than 90% (byweight), usually less than 60% and more usually less than 50% of thecomposition is made up of other polypeptides.

The antibody can be of any isotype (e.g., IgA, IgG, IgM i.e. an α, γ orμ heavy chain). Within the IgG isotype, the antibodies may be IgG1,IgG2, IgG3 or IgG4 subclass. The antibodies may have a κ or a λ lightchain.

Production of Antibodies

Antibodies for use in the invention can be made by any method known inthe art. For example, the general methodology for making monoclonalantibodies using hybridoma technology is well known (Kohler, G. andMilstein, C,. 1975; Kozbar et al. 1983). In one embodiment, thealternative, improved EBV immortalization method described inWO2004/076677 is used.

Using the method described in WO 2004/076677, B cells producing theantibody of the invention can be transformed with EBV and a polyclonal Bcell activator. Additional stimulants of cellular growth anddifferentiation may optionally be added during the transformation stepto further enhance the efficiency. These stimulants may be cytokinessuch as IL-2 and IL-15. In one aspect, IL-2 is added during theimmortalization step to further improve the efficiency ofimmortalization, but its use is not essential. The immortalized B cellsproduced using these methods can then be cultured using methods known inthe art and antibodies isolated therefrom.

Using the method described in WO 2010/046775, plasma cells can becultured in limited numbers, or as single plasma cells in microwellculture plates. Antibodies can be isolated from the plasma cellcultures. Further, from the plasma cell cultures, RNA can be extractedand PCR can be performed using methods known in the art. The VH and VLgene regions of the antibodies can be amplified by RT-PCR, sequenced andcloned into an expression vector that is then transfected into HEK293Tcells or other host cells. The cloning of nucleic acid in expressionvectors, the transfection of host cells, the culture of the transfectedhost cells and the isolation of the produced antibody can be done usingany methods known to one of skill in the art.

The antibodies may be further purified, if desired, using filtration,centrifugation and various chromatographic methods such as HPLC oraffinity chromatography. Techniques for purification of antibodies,e.g., monoclonal antibodies, including techniques for producingpharmaceutical-grade antibodies, are well known in the art.

Fragments of the antibodies can be obtained from the antibodies bymethods that include digestion with enzymes, such as pepsin or papain,and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, fragments of the antibodies can be obtained by cloningand expression of part of the sequences of the heavy or light chains.Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fv fragments. Theinvention also encompasses the use of single-chain Fv fragments (scFv)derived from the heavy and light chains of an antibody of the invention.For example, the invention includes the use of a scFv comprising theCDRs from a neutralizing antibody. Also included are the use of heavy orlight chain monomers and dimers, single domain heavy chain antibodies,single domain light chain antibodies, as well as single chainantibodies, e.g., single chain Fv in which the heavy and light chainvariable domains are joined by a peptide linker.

Antibody fragments may impart monovalent or multivalent interactions andbe contained in a variety of structures as described above. Forinstance, scFv molecules may be synthesized to create a trivalent“triabody” or a tetravalent “tetrabody.” The scFv molecules may includea domain of the Fc region resulting in bivalent minibodies. Additionalmolecules for use in the invention include, but are not limited to,bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies.

Standard techniques of molecular biology may be used to prepare DNAsequences encoding the antibodies or antibody fragments. Desired DNAsequences may be synthesized completely or in part using oligonucleotidesynthesis techniques. Site-directed mutagenesis and polymerase chainreaction (PCR) techniques may be used as appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example, E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)2 fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e.g., mammalian, host cell expression systems may be usedfor production of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include, but are not limitedto, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

Alternatively, antibodies for use in the invention may be produced by(i) expressing a nucleic acid sequence encoding an antibody of interestin a host cell, and (ii) isolating the expressed antibody product.Additionally, the method may include purifying the isolated antibody.

Transformed B cells and cultured plasma cells may be screened for thoseproducing antibodies of the desired specificity or function. Thescreening step may be carried out by any immunoassay, e.g., ELISA, bystaining of tissues or cells (including transfected cells), byneutralization assay or by one of a number of other methods known in theart for identifying desired specificity or function.

Individual transformed B cell clones may be produced from the positivetransformed B cell culture. The cloning step for separating individualclones from the mixture of positive cells may be carried out usinglimiting dilution, micromanipulation, single cell deposition by cellsorting or other methods known in the art.

Nucleic acid from the cultured plasma cells can be isolated, cloned andexpressed in HEK293T cells or other known host cells using methods knownin the art.

Pharmaceutical Compositions

The invention provides a pharmaceutical composition comprising anantibody and/or an antigen, as described above. The pharmaceuticalcomposition may also contain a pharmaceutically acceptable carrier,diluent or excipient. Although the carrier or excipient may facilitateadministration, it should not itself induce the production of antibodiesharmful to the individual receiving the composition, nor should it betoxic. Suitable carriers may be large, slowly metabolized macromoleculessuch as proteins, polypeptides, liposomes, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymersand inactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the subject.

Within the scope of the invention are compositions present in severalforms of administration; the forms include, but are not limited to,those forms suitable for parenteral administration, e.g., by injectionor infusion, for example by bolus injection or continuous infusion.Where the product is for injection or infusion, it may take the form ofa suspension, solution or emulsion in an oily or aqueous vehicle and itmay contain formulatory agents, such as suspending, preservative,stabilizing and/or dispersing agents. Alternatively, the antibodymolecule may be in dry form, for reconstitution before use with anappropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. In one embodiment the compositions are adaptedfor administration to mammalian, e.g., human subjects.

The pharmaceutical compositions of this invention may he administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intranasal, enteral, sublingual, intravaginal or rectalroutes. Hyposprays may also be used to administer the pharmaceuticalcompositions of the invention. Typically, the therapeutic compositionsmay be prepared as injectables, either as liquid solutions orsuspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule. Knownantibody-based pharmaceuticals provide guidance relating to frequency ofadministration e.g., whether a pharmaceutical should be delivered daily,weekly, monthly, etc. Frequency and dosage may also depend on theseverity of symptoms.

Compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g., a lyophilized composition, like Synagis™ and Herceptin™,for reconstitution with sterile water containing a preservative). Thecomposition may be prepared for topical administration e.g., as anointment, cream or powder. The composition may be prepared for oraladministration e.g., as a tablet or capsule, as a spray, or as a syrup(optionally flavoured). The composition may be prepared for pulmonaryadministration e.g., as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g., asdrops. The composition may be in kit form, designed such that a combinedcomposition is reconstituted just prior to administration to a subject.For example, a lyophilized antibody can be provided in kit form withsterile water or a sterile buffer.

The compositions of the invention elicit both a cell mediated immuneresponse as well as a humoral immune response.

Medical Treatments and Uses

The antibodies, antigens, and compositions disclosed herein can be usedin the present improved methods for generating, stimulating, oreliciting an active vaccine or immune response in a subject,ameliorating or eliminating the severity of an infection in a subject,or limiting infection of a subject by a pathogen, and in the preparationof medicaments for these purposes.

General

As used herein, the term antibody includes a complete antibody as wellas antigen binding fragments thereof as described above. The terms“antigen binding fragment,” “fragment,” and “antibody fragment” are usedinterchangeably to refer to any fragment of an antibody of the inventionthat retains the antigen-binding activity of the antibody. Examples ofantibody fragments include, but are not limited to, a single chainantibody, Fab, Fab′, F(ab′)₂, Fv or scFv. Further, the term “antibody”as used herein includes both antibodies and antigen binding fragmentsthereof.

As used herein, a “neutralizing antibody” is one that can neutralize,i.e., prevent, inhibit, reduce, impede or interfere with, the ability ofa pathogen to initiate and/or perpetuate an infection in a host. Theterms “neutralizing antibody” and “an antibody that neutralizes” or“antibodies that neutralize” are used interchangeably herein. Theseantibodies can be used alone, or in combination, as prophylactic ortherapeutic agents upon appropriate formulation, in association withactive vaccination, as a diagnostic tool, or as a production tool asdescribed herein.

As used herein, an “equivalent” with respect to an infectious pathogenis a pathogen with the same taxonomic description and bearing therelevant antigenic molecules.

The term “comprising” encompasses “including” e.g., a composition“comprising” X may include something additional to X, e.g., X+Y.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means x±10%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder,”“infection” and “condition” (as in medical condition), in that allreflect an abnormal condition of the human or animal body or of one ofits parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life.

As used herein, reference to “treatment” of a subject or patient isintended to include prevention, prophylaxis, attenuation, ameliorationand therapy. The terms “subject” or “patient” are used interchangeablyherein to mean all mammals including humans. Examples of subjectsinclude humans, cows, dogs, cats, horses, goats, sheep, pigs, andrabbits. In one embodiment, the patient is a human.

EXAMPLES

Exemplary embodiments of the present invention are provided in thefollowing examples. The following examples are presented only by way ofillustration and to assist one of ordinary skill in using the invention.The examples are not intended in any way to otherwise limit the scope ofthe invention.

Example 1

The purpose of this experiment is to evaluate whether the administrationof a monoclonal neutralizing antibody before infection can influence theendogenous immune response to Influenza A virus infection. Thisexperiment shows the differing levels of protection conferred to mice towhich is administered influenza A virus and differing levels of a potentneutralizing antibody, FI6 (see WO 2010/010466).

A neutralizing antibody (FI6) was administered intravenously to mice atdifferent doses (0.1, 1, and 10 mg/kg) 4 hours before intranasalinfection with a sub-lethal dose of Influenza A H1N1 A/Puerto Rico/8/34virus. Body weight loss and survival were followed for 14 days afterinfection (FIG. 1).

Mice receiving 10 mg/kg FI6 showed no body weight loss and no symptoms(FIG. 1D). Mice receiving 1 mg/kg FI6 showed only a very modest bodyweight loss without measurable symptoms (ruffled fur and/or respiratorydistress) (FIG. 1C), while those receiving 0.1 mg/kg FI6 showed asubstantial body weight loss, morbidity, and mortality comparable to thecontrol group of mice receiving phosphate saline buffer only (FIG.1A-B).

The immune response in these animals was assessed in serum 25 days afterinfection by measuring the presence of mouse IgG antibodies able to bindor neutralize the challenge virus (EC50 and IC50 values). Mice treatedwith 1 mg/kg FI6 showed an immune response significantly greater thanthat in animals treated with 10 mg/kg antibody, and comparable to theantibody response observed in infected untreated animals (FIG. 2).

These results demonstrate that the administration of a monoclonalneutralizing antibody at an appropriately selected dose is able tocontain virus replication and can be instrumental in eliciting, in theabsence of severe morbidity, a robust immune response against thatspecific pathogen.

Example 2

The purpose of this experiment is to evaluate whether the administrationof a monoclonal antibody after infection can influence the endogenousimmune response to rabies virus infection.

Two rabies virus neutralizing antibodies were administered as a cocktailto Syrian hamsters one or five days after intramuscular infection with alethal dose of rabies virus. This treatment contained viral spreadinginto the CNS, decreasing the severity of symptoms and leading to thesurvival of all treated animals (FIG. 3A).

Importantly, the antibody treatment did not prevent the production ofhigh levels of endogenous antibodies against the G protein (which is themajor target of neutralizing antibodies) (FIG. 3B). The endogenousantibodies, along with T cell responses, might be required to suppressrabies virus spreading and neurologic symptoms.

These results demonstrate that the administration of neutralizingantibodies after infection with a lethal virus not only provideshort-term passive protection to the animal but also elicits a robustlong-term active endogenous immune response able to further containvirus spreading and related neurologic symptoms.

Each of the references cited herein is incorporated herein by referencein its entirety.

What is claimed is:
 1. A method of generating an active immune responsein a subject, the method comprising administering to the subject aneffective amount of a rabies-neutralizing monoclonal antibodyconcurrently or sequentially with a rabies antigen.
 2. The method ofclaim 1, wherein: (a) the rabies antigen is administered between about 5minutes to about 3 weeks following administration of the monoclonalantibody; or (b) the monoclonal antibody is administered between about 5minutes to about 3 weeks following administration of the rabies antigen.3. The method of claim 1, wherein the monoclonal antibody neutralizesinfectivity of the rabies virus.
 4. The method of claim 1, wherein therabies antigen is an attenuated virus or a recombinant vaccine.
 5. Themethod of claim 2, wherein: (a) the rabies antigen is administeredbetween about 1 hour to about 3 weeks following administration of themonoclonal antibody; or (b) the monoclonal antibody is administeredbetween about 1 hour to about 3 weeks following administration of therabies antigen.
 6. The method of claim 1, wherein administering themonoclonal antibody comprises administering two rabies-neutralizingmonoclonal antibodies to the subject.
 7. The method of claim 1, whereinthe subject is immunologically protected from the rabies antigen.
 8. Themethod of claim 1, wherein the response is persistent and protective. 9.The method of claim 1, wherein the monoclonal antibody and the rabiesantigen are administered by the same route or by different routes. 10.The method of claim 9, wherein the route(s) are selected from the groupconsisting of oral, dermal, topical, intranasal, intramuscular,intravenous, intrathecal, intravaginal, intravitreal, and subcutaneous.11. A method of ameliorating or eliminating the severity of an existingrabies infection or of limiting an existing rabies infection in asubject, comprising administering to the subject an effective amount ofan antibody concurrently or sequentially with a rabies antigen, whereinthe antibody neutralizes infectivity of the rabies virus.
 12. The methodof claim 11, wherein: (a) the rabies antigen is administered betweenabout 5 minutes to about 3 weeks following administration of theantibody, or; (b) the antibody is administered between about 5 minutesto about 3 weeks following administration of the rabies antigen.
 13. Themethod of claim 11, wherein the rabies antigen is an attenuated virus ofthe rabies virus causing the infection, or is a recombinant vaccinederived from the rabies virus causing the infection.
 14. The method ofclaim 12, wherein: (a) the rabies antigen is administered between about1 hour to about 3 weeks following administration of the antibody; or (b)the antibody is administered between about 1 hour to about 3 weeksfollowing administration of the rabies antigen.
 15. The method of claim11, wherein the subject is immunologically protected from the rabiesvirus.
 16. The method of claim 11, wherein the antibody and the rabiesantigen are administered by the same route or by different routes. 17.The method of claim 16, wherein the route(s) are selected from the groupconsisting of oral, dermal, topical, intranasal, intramuscular,intravenous, intrathecal, intravaginal, intravitreal, and subcutaneous.18. The method of claim 11, wherein the antibody enhances the subject'sendogenous immunological response to the rabies virus and neutralizesinfectivity of the rabies virus.
 19. The method of claim 11, wherein theantibody is a monoclonal antibody.
 20. The method of claim 11, whereinadministering the antibody comprises administering to the subject twoantibodies that neutralize infectivity of the rabies virus.